Lokesh Saravanan

Advances and Emerging Mechanisms of 3-D Printed Scaffolds in Bone Tissue Engineering Lokesh Saravanan, Moharam Sabira A, Saranya Srinivasan, Ashok Kumar Pandurangan* School of Life Sciences, B. S. Abdur Rahman Crescent Institute of Science and Technology Vandalur, Chennai Corresponding author Mail ID:-Abstract Bone Tissue Engineering is a rapidly evolving field within biomedical research, focused on developing innovative biomaterials, cells, and growth factors to address damage or injury in bone tissues. Effective bone regeneration relies heavily on maintaining bone homeostasis and facilitating remodelling, areas currently under extensive investigation through various advanced techniques and technologies. Among these approaches, 3D Bio-Printing, a technique rooted in regenerative medicine, stands out for its ability to fabricate three-dimensional scaffolds, tissues, and even organs. This process addresses limitations commonly encountered with traditional methods like lyophilization and solvent casting, such as non-uniform pore sizes and structural inconsistencies. Research has highlighted the pivotal role of diverse polymers and their functionalities in creating bio-printed scaffolds tailored for targeted bone regeneration applications. This review examines the details of 3D Bio-Printing, detailing the methodologies, polymeric materials, and their molecular mechanisms in bone tissue repair. Additionally, it explores the broader clinical implications and potential future directions for 3D-printed scaffolds in advancing bone regeneration strategies. Keywords: 3-D Bio-Printing, Biomaterials, Molecular mechanisms, Bone Regeneration 1. Introduction Bone as an important part in our body as it serves various functions such as, offering structural support, protects the vital internal organs, stores minerals such as calcium and phosphorus and supports the immune system by housing the stem cells required for differentiation. The problem with regenerating bone tissue is a very complex biological process because they require great intrinsic mechanical and physiological demands on the skeletal system[1]. Clinical approaches for the management of bone defects conventionally involve autografts, allografts, and xenografts[2]. However, these options present a number of limitations related to donor-site morbidity, immune rejection, availability of limited grafts, and high complication rate following surgery[3]. These challenges have driven the emergence of Bone Tissue Engineering (BTE), which is a multidisciplinary field focused on scaffold design and construction that able to mimic natural bone ECM and thus providing a framework through which the innate body repair mechanisms act [4],[5]. Scaffold is a type of biomaterials that is crucial for bone regeneration as it provides a temporary framework for the infiltration of osteoprogenitor cells to proliferate and differentiate. Some of the properties of the scaffolds includes biocompatibility, osteo-conductivity, mechanical strength, and biodegradability [6],[7]. It should have the ability to promote vascularization and angiogenesis, which refers to the provision of nutrition and the removal of wastes, as they are critical for bone healing process [8]. Traditional scaffold fabrication techniques like freeze-drying and solvent casting typically have some challenges in pore size control, through the formation of inconsistent pore size and unbalancing in the optimum porosity range , which replicate the complicate geometry of the scaffold that affects its mechanical strength and durability of the scaffold [9], [10]. The invention of 3D printing techniques revolutionized scaffold fabrication due to its high accuracy, customizable and reproducible. They have the ability to mimic native bone architecture [11]. With 3D printing, researchers can tailor the design of scaffolds for specific anatomical needs, optimize porosity for nutrient diffusion, and fabricate them with properties matching the mechanical environment at the targeted bone site[12]. These characteristics make 3D printing even more attractive for conditions with large bone defects or when complex bone shapes must be achieved [13]. In addition to the biocompatible polymers such as gelatin, alginate, chitosan and collagen, the bio-ceramics such as hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), bioactive glass enhance the calcium absorption of the biomaterials [14]. Thus, the composite materials fabricated using 3-D Bioprinting, is shown to have enhance controlled drug release. This review provides a comprehensive overview of advancements in 3D printed scaffolds in bone tissue engineering, detailing the roles of stem cells, growth factors, and immune cells in the scaffold microenvironment, which collectively drive bone regeneration. It highlights state-of-the-art 3D printing techniques and also addresses clinical challenges like scaffold degradation, mechanical failures, and vascular integration, alongside regulatory and ethical considerations, thereby providing future prospectives in bone tissue regeneration. 2.Advanced 3D printing Technologies for scaffold fabrication Advanced 3D printing technology is preferred over conventional methods in tissue engineering and regenerative medicine. Traditional techniques, such as solvent casting, gas foaming, and freeze-drying, often fabricate scaffolds with inconsistent pore structures but possess challenges in adjusting the morphology and pore sizes [15]. In contrast, advanced printing allows for precise control over the architecture of the scaffold. This capability enables researchers to create highly complex, biomimetic structures with tailored porosity and features that facilitate cell migration, nutrient diffusion, and waste excretion. Advanced techniques including stereolithography (SLA) and laser sintering can closely replicate the intricate geometry of the native tissues [16]. In addition to precision, this advanced technology supports a wide range of biomaterials, including biodegradable polymers, hydrogels, and composites that can be incorporated with bioactive components to enhance cell attachment and proliferation [17]. Achieving the necessary customization for developing scaffolds for different tissues, such as bone or cartilage, is challenging and requires proper mechanical and biological characteristics. To address this issue, advanced technologies like inkjet printing, especially bioprinting, allow for the direct incorporation of cells during fabrication. This enables the creation of cell-laden scaffolds that closely resemble functional tissues [18]. The ability to directly integrate living cells into the scaffolds makes advanced 3D printing an ideal choice for producing functional structures for both in vitro and clinical studies. 2.1. Extrusion-based bioprinting Pressure or Extrusion-based technology is an increasingly prominent platform known for its versatility in printing various biological materials such as tissues and organ models. The initial study concerning Extrusion-based Bioprinting (EBB) focused on the deposition of living cells through a plotting technique, in which hydrogel bio-ink is extruded and bio-plotted into a liquid environment, yielding high mechanical strength and promoting cell growth [19]. The EBB method integrates a fluid-dispensing mechanism with an automated robotic system for extrusion and bioprinting, respectively. During the bioprinting process, bioink is dispensed by a delivery system managed by the software, allowing for precise placement of cells within cylindrical filaments that create customized 3D structures (figure 1) [20]. This rapid fabrication technique enhances structural integrity through the continuous deposition of filaments. Additionally, this approach can incorporate computer programs such as computer-aided design (CAD) software, enabling users to upload a CAD file for automatic structure printing [21]. The CAD file can be derived from medical images such as MRI and CT scans or created based on specific design requirements. Micro-extrusion techniques accommodate a wide range of fluid characteristics that are suitable for the procedure, using a diverse selection of biocompatible materials documented in research. Materials with viscosities ranging from 30 mPa/s to over 6 × 107 mPa/s have been shown to work well with micro-extrusion bioprinters; typically, higher-viscosity materials enhance structural support for the printed objects, while lower-viscosity materials help maintain cell viability and function [22]. For micro-extrusion bioprinting, flow at physiologically compatible temperature (35-40°C) but interlinked temperature between process is also beneficial in bioprinting. Materials exhibiting shear-thinning behaviour are frequently utilized in micro-extrusion applications. This Non-Newtonian behaviour results in the reduction of viscosity, as the shear rate increases [23]. The elevated shear rates present at the nozzle during bio-fabrication enable these materials to flow through the nozzle, and when deposited, the shear rate drops, leading to a significant viscosity increase. The high resolution of micro-extrusion systems allows bioprinters to fabricate intricate structures designed via CAD software and enables the arrangement of various cell types. The primary advantage of micro-extrusion bioprinting technology is its capability to deposit extremely high cell densities. 2.2. Inkjet Bioprinting Inkjet printing employs a dispensing unit to place small quantities of liquid material onto a surface (figure 1). The primary benefit of this technology is the capacity to precisely manage tiny particles, which can be formed into specific droplets [24]. Furthermore, this technology is incorporated for the manufacturing of medical devices and economical ink formulations that are also highly adaptable for prototyping since inkjet printing is a maskless method, making it easy to modify desired patterns by adjusting the digital printing files [25]. There are two main approaches to this printing: Piezoelectric inkjet printing and Thermal inkjet technology. In Piezoelectric inkjet printing, the formation of drops is facilitated by a piezoelectric component linked to the printing nozzle. By applying appropriate voltage pulses, a pressure wave is created within the nozzle, leading to the ejection of ink in a stream that subsequently separates from the nozzle. The production of well-defined droplets of the printing material heavily relies on the balance between the surface tension and the viscosity of the expelled liquid [26]. The alternative approach involves temperature, which is prevalent in consumer and lab inkjet printing devices, employing a thermal inkjet system, where droplets are expelled from the nozzle by generating an air bubble on the surface of a tiny heating element positioned near the jetting nozzle [25]. When a current pulse is applied, the heater heats up rapidly in under a few microseconds and transfers heat to the ink. This causes the ink temperature to reach a critical threshold, resulting in bubble formation. Once the bubble collapses on the heating element’s surface, the ink stream separates from the nozzle, creating the droplet that is then ejected toward the surface [26]. 2.3. Selective Laser Sintering (SLS) SLS falls under the category of Powder Bed Fusion as it is recognized by the ASTM (American Society for Testing Materials). This process involves the creation of objects by fusing powder particles with energy provided by a laser. This technique of additive manufacturing offers several advantages, including high resolution, the possibility of reusing the powder, and no need for pre-processing [27]. In powder-bed fusion, a fine layer of powder is spread across the build platform, and a laser selectively fuses the particles based on a 3D model. This layer-by-layer process continues, with the unfused powder serving as support, until the entire object is constructed. Once printing is complete, the excess powder is removed to reveal the final product. When the laser strikes the powder, it heats the particles to their melting point or close to it, causing them to bond together [28]. This process, known as “sintering,” results in the formation of a solid layer, and after each layer is formed, the platform is lowered slightly to make space for the next layer (figure 1). As subsequent layers are sintered on top of the previous layers, the structure gradually takes shape, with the unsintered powder that surrounds it. This provides support for the overhangs and intricate designs [29]. This method not only facilitates the creation of highly detailed and structurally sound scaffolds but also reduces waste, as the powder which is sintered is only utilized, while the excess powder can be recycled. SLS is an advantageous technology, particularly useful for manufacturing scaffolds designed for load-bearing uses, due to its capability to create strong, dense components with high mechanical strength. In the field of bioprinting, SLS is frequently used to produce scaffolds from materials such as polycaprolactone (PCL) and polyether ether ketone (PEEK), which are known for their biocompatibility and durability [30]. 2.4 Stereolithography (SLA) Stereolithography is the first laser-based printing technology. The term "SLA" was coined by Chuck Hull in the 1980s. There are two main types of SLA: Laser-based SLA and Digital light projection [16]. In the laser-based method, products are fabricated using a bottom-up approach, where a computer-controlled laser beam system directs UV light through the transparent bottom of a vat filled with photosensitive resin. This stereolithographic technique has several unique features, including fine resolution that allows for the creation of complex structures. Additionally, the printing process generates minimal heat, which helps to avoid thermal degradation of drugs. As a result, SLA is particularly beneficial for thermo-labile drugs [31]. The basic principle of Stereolithography (SLA) involves passing ultraviolet (UV) radiation through a photosensitive resin to produce photopolymerized structures. The core mechanism of this process is photopolymerization. When UV radiation is directed at the resin, a thin layer solidifies on the surface, which is then immersed in a liquid bath (figure 1). The movable platform plays a crucial role in this setup. Once the 3D object is formed, all parts of the machine are cleaned using a solvent. Additionally, pharmaceutical formulations that contain a higher amount of water show an increased effect on drug release when exposed to irradiation, with the release rate rising from 27% to 95% [32]. Stereolithography relies on photo-polymerization, where the controlled solidification of liquid resin takes place. A container filled with liquid resin (photopolymer) is positioned on the main platform, which remains fixed. An elevator featuring a movable platform is situated within the vessel, connected to the main platform. At the beginning, the movable platform is raised close to the top of the liquid resin photopolymer [33]. Following one cycle of UV laser exposure, a new layer of photopolymerized material is created, with the thickness of the subsequent layer determined by lowering the platform. The creation of the 3D object occurs through a series of repeated laser treatments. Figure 1: The different methods of 3-D Bio-Printing of a scaffold (A)extrusion-based bioprinting (B)inkjet bioprinting (C)selective laser sintering (D)Stereolithography approach 3. Bioactive materials in 3D printed scaffolds The chronic abnormalities in bone are often treated with bone grafts or bone replacement materials, as bone is the most transplanted tissue worldwide. Beside using bioactive materials, the advanced 3D printed scaffolds is crucial for promoting bone regeneration (figure 2). Certain types of materials, including hydrogels, metals, ceramics, and polymers, have limitations in replicating the properties of bone. However, their performance improves significantly when combined to form composite scaffolds [34]. Due to the similar osteo-conductivity and biocompatibility to bone, hydroxyapatite (HA) has been studied as a potential bone substitute. But it is brittle and different to process into complex shapes [35]. HA supports cell attachment, proliferation, and differentiation. To address HA’s inherent brittleness, it is combined with polymers and ceramics to improve its mechanical properties. It is also engineered as drug carriers, where they release antibodies or growth factors directly at the site of implantation to promote bone regeneration [36]. One of the combinations is Alginate/HA, this combination enhances biomineralization, crucial for dental pulp and bone regeneration. The nanocomposite structure of these scaffolds provides porous matrix that support nutrient diffusion and cell infiltration. Alginate/HA scaffolds exhibit tunable degradation rates, allowing scaffold resorption as the new tissue forms [37]. The HA-TCP scaffold is also known to provide an osteoconductive matrix, supporting bone cell attachment and proliferation, its porous structure allows for efficient nutrient exchange and vascularization. The combination of HA- TCP with rhBMP-2 significantly boosts the bone formation during distraction osteogenesis, which involves bone elongation [38]. Calcium phosphate and glass composite coatings were studied to enhance zirconia’s biocompatibility. The coating promotes osseointegration by mimicking bone mineral content. These coatings increase the surface roughness of zirconia, which aids in cell attachment and bone cell response. It also enhances corrosion resistance and durability, which is essential for the long-term stability of implants [39]. The most widely used and effective synthetic bone graft alternative is β- tricalcium phosphate (β – TCP). The significant properties of β - TCP are osteo-conductivity and osteo-inductivity [40]. Some studies shown that the β- TCP is coated with PLGA (co-poly lactic acid/glycolic acid) to improve the mechanical strength and biological performance. The PLGA/β-TCP scaffolds maintain high porosity and tissue compatibility [41]. Figure 2: 3-D Bio-Printed scaffolds have been studied to promote bone regeneration, when implanted at the site of injury/damage 3.1 Biomaterials and Copolymers in 3D-Printed Scaffolds S.no Polymers Co-polymers Application References 1. Chitosan Poly (lactic acid) (PLA) Improves osteo-conductivity, biocompatibility, and mechanical qualities. [42] 2. Collagen Poly (lactic-co-glycolic acid) (PLGA) Stimulates osteogenic development in bone scaffolds and mimics extracellular matrix. [43] 3. Silk fibroin Polycaprolactone (PCL) Imparts strength and flexibility to load-bearing bone deformities. [44] 4. Alginate Polyethylene glycol (PEG) Develops hydrogels for nutrient diffusion and osteoblast adhesion. [45] 5. Hyaluronic acid Poly (N-iso propylacrylamide) (PNIPAAm) Stem cell encapsulation is supported by thermoresponsive hydrogel. [46] 6. Gelatin Poly (propylene fumarate) (PPF) Improves mechanical stability and osteo-inductive qualities. [47] 7. Fibrin Poly (vinyl alcohol) (PVA) Promotes osteogenic differentiation and aids in cell encapsulation. [48] 8. Cellulose (Bacterial) Polycaprolactone (PCL) Bone scaffolds strengthened by nanostructures [49] 9. Starch Poly (lactic acid) (PLA) Bioactive filler in scaffolds of composite bone [50] 10. Carrageenan Polycaprolactone (PCL) Increasing the scaffold’s structural integrity [51] Table 1: The list of polymers and co-polymers as a 3-D Bio-printed scaffolds and their applications in Bone targeted bone deformities. 3.2 Polymers and Bio-ceramics in 3D-Printed Scaffolds S.no Polymer Bio ceramic Application Reference 1. Gelatin Hydroxyapatite (HA) Supporting bone growth and osteointegration [52] 2. PCL Tricalcium phosphate (TCP) Enhancing osteoconductive and mechanical qualities [53] 3. PLA Bioactive glass Increasing osteogenesis and bioactivity [54] 4. PLGA Biphasic calcium phosphate Bone regeneration through balanced resorption [55] 5. Polyethylene glycol HA Hydrogel support structures for cell proliferation [56] 6. Polyurethane (PU) TCP Applications of load-bearing bone tissue [57] 7. Polyhydroxybutyrate HA Strong, biocompatible scaffolds for major defects [58] 8. PVA Bioactive glass Hydrophilic scaffolds to improve cell adhesion [59] 9. Polydioxanone HA Resorbable scaffolds for the healing of bone defects [60] 10. Poly (propylene fumarate) Carbonated Apatite Mimics the mineral composition of bone to facilitate scaffold incorporation. [61] Table 2: The list of Polymers and Bio-ceramics as a 3-D Bio-printed scaffolds and their applications in Bone tissue engineering. 4.Emerging mechanisms of 3D printed Scaffolds in bone regeneration The bone regeneration occurs through a complex biological procedures controlled by the cells and their signalling molecules through the interactions between them in the extracellular matrix they produce [62]. Recent progress in 3D printing techniques allows designing scaffolds that actively contribute to bone healing by promoting osteogenesis, facilitating angiogenesis and modulating immune responses [63]. Such mechanisms are paramount relevance in optimizing scaffold integration and functional recovery in BTE. Below, we listed the major emerging mechanisms that are currently under active investigation in 3D-printed scaffolds and their contribution to improve clinical outcomes [64]. 4.1 Osteo-induction and Stem Cell Differentiation Osteo-induction is the induction of mesenchymal progenitor cells to bone-forming osteoblasts and forms one of the basic principles of bone healing. Osteo-inductive properties of 3D-printed scaffolds may be considered fabricating through the incorporation of bioactive factors and/or ECM components, enhancing the differentiation of stem cells in order to achieve osteogenesis [65]. 4.1.1 Growth Factor Integration: It has been indicated that the addition of such osteo-inductive growth factors such as BMP-2 and TGF-β to the scaffolds, enhanced the differentiation of MSCs into osteoblasts [66]. For example, controlled release systems, in which growth factors are encapsulated within biodegradable microspheres or delivered within hydrogel layers, have been shown to have maximal impacts on osteogenic outcomes due to sustained delivery and retention of the growth factor at the defect site [67]. 4.1.2 ECM Protein Functionalization: There has been proof that functionalization of scaffolds with ECM proteins like collagen and fibronectin enhances the cell adhesion, proliferation, and osteogenic differentiation. These proteins act as attachment sites for the cells and also activate integrin-mediated signalling pathways, thereby promoting maturity in osteoblasts [68], [69]. 4.1.3 Peptide and Small Molecule Modifications: Small peptides, including RGD [Arg-Gly-Asp], and other molecules specific to osteogenic induction, such as strontium have been integrated with the biomaterials, as a strategy to further enhance osteo-induction [70], [71]. The application of peptides introduces specific binding sites for integrins of MSCs, and therefore cell attachment is enabled with further signalling along the pathways connected to osteoblastic lineage differentiation. 4.2 Angiogenesis and Vascular Integration Scaffolds must support vascularization for effective bone regeneration, due to the need for blood vessels to supply the oxygen and nutrients needed to sustain tissue regeneration and to remove waste products. New scaffold designs now aim at triggering endothelial cell proliferation and also the formation of new blood vessels inside the scaffold structure [72]. 4.2.1 Porous Structure and Interconnectivity: In order to offer effective encouragement of endothelial cell migration for vascular network development, porosity and interconnectivity of 3D-printed scaffolds should be optimized. Based on certain literature findings, the pore size of 200-500 µm is considered to be optimal for vascular invasion in bone scaffolds [12], [73]. Advanced bioprinting techniques can make controlled microarchitectures possible. This allows predesigned vascular channels that accelerate angiogenesis in-vivo to be achieved [74]. 4.2.2 Angiogenic Factor Incorporation: The most commonly used angiogenic agents are VEGF, FGF, and PDGF, which are incorporated into the scaffold to encourage blood vessel growth. Of these, VEGF seems to be the better one for the induction of endothelial cell differentiation and neovascularization within the scaffold matrix [75]. Controlled release mechanisms and hydrogels within the scaffold structure allow for the gradual and localized release of VEGF, thus optimizing vascularization outcomes. Co-culturing with endothelial cells is one more approach wherein co-printing technique that introduce endothelial cells together with osteogenic cells realize the establishment of vascular and bone networks all at once in support of more functional tissue integration. This co-culture approach has been shown to increase the survival rate of cells and functionality of scaffolds, especially in large bone defects where vascularization should take place rapidly [76]. 4.3 Immune Modulation and Inflammatory Response Immune modulation is a developing mechanism important for scaffold biocompatibility and functional integration. The immune response itself is important in tissue healing and scaffold modelling. Modulation of the immune environment within the scaffold can favour a pro-healing phenotype in the immune cells to reduce fibrosis and enable better integration of the scaffold [77]. 4.3.1 Polarization of Macrophages: Macrophages, in particular the M2 phenotype, plays a crucial role in wound healing and tissue repair. Immuno-modulatory agent-drug scaffold designs by incorporating IL-10 and IL-4, which polarize macrophages towards an anti-inflammatory M2 phenotype. This polarization is constructive for having a better environment for bone healing, since the production of pro-inflammatory cytokines is reduced and growth factor secretion is increased [78], [79]. 4.3.2 Anti-inflammatory Coatings and Drug Delivery: The addition of anti-inflammatory agents such as dexamethasone or curcumin within the scaffold matrices helps to control inflammation at the implant site for minimal risk of chronic inflammation and fibrosis [80], [81]. These agents are often embedded within nanocarriers or loaded onto scaffold surfaces for controlled, localized release. 4.3.3 Bacterial Infection Control: The prevention of bacterial infection is a paramount in the load-bearing applications. Modifications on the scaffold surface with antibacterial agents such as silver nanoparticles or chitosan will help in controlling inflammation and prevention of biofilm formation, hence reducing the infection risks dramatically while preventing inflammation [82]. Mechanism Key Materials/Factors Function Observed Outcomes in Preclinical and invitro studies References Osteo-induction BMP-2 By activating the Smad signaling pathway induces the MSC differentiation into osteoblasts Improved, bone formation, increased mineralized tissue in critical-sized defects [65] TGF-β Controls the synthesis of extracellular matrix and the maturation of osteoblasts, which promotes MSC proliferation and osteogenic differentiation. Enhanced scaffold integration, increased ECM deposition, and accelerated fracture healing. [66], [67] Collagen Offers bioactive binding sites and structural support for signaling and cell attachment. Enhanced osteogenic differentiation, scaffold biocompatibility, and cell adhesion. [68], [69] ,RGD Peptides Mimics integrin-binding domains to encourage osteoblast differentiation and cell adhesion. ncreased osteoblast lineage specificity and cellular proliferation. [69], [70] Angiogenesis VEGF Encourages the migration, proliferation, and creation of new blood vessels by activating the VEGF receptor. nhanced oxygen/nutrient delivery and increased vascularization inside scaffold matrices. [75] FGF Promotes the growth of endothelial cells and strengthens angiogenic signaling pathways. Enhanced bone regeneration and accelerated vascular network creation in vivo. [76] Immune Modulation IL-10 Suppresses pro-inflammatory cytokines and encourages the phenotype of M2 macrophages that are pro-healing. Enhanced scaffold integration, less fibrosis, and quicker bone regeneration. [77], [78] IL-4 Maximizes anti-inflammatory responses by polarizing macrophages toward the M2 phenotype. Improved tissue repair decreases the levels of inflammatory cytokines. [79] Dexamethasone Promotes osteogenic activity and suppresses inflammation by blocking NF-κB signaling. Less long-term inflammation and more bone growth during the inflammatory conditions. [80] Silver Nanoparticles Reduces inflammation and encourages tissue regeneration while offering antibacterial qualities. Enhanced healing effectiveness, decreased biofilm formation, and decreased infection risk. [82] Table 4: The involvement of growth factors and the bioactive particles and their mechanism involved in bone regeneration applications 5. Bioprinting and cell laden scaffolds Bioprinting is the process of creating 3-D structures, that mimics the tissues and organs using bioinks. These bioinks are made up of cells, nutrients and sometimes growth factors, which makes the mimicking the tissues after printing. The main goal of bioprinting is to build tissues, for their biomedical application like repairing the damaged tissues and organs. One of the main challenges in bioprinting is the maintenance of uniformity of the cells in the bioink. That’s where cell laden scaffolds comes in, as they provide a supportive matrix within the bioink to hold cells in place, helps to maintain an uniform distribution [83]. Among the diverse approaches of bioprinting, each of them offers distinct advantages in creating precise, cell-laden structures tailored for specific tissue engineering applications. Inkjet bioprinting, which is based on the conventional 2-D desktop printing, produces 3-D structures by using bioink droplets. Cell-containing bioinks are sprayed in precise droplets to create defined patterns [84]. This technique is likely to be used often for its effectiveness in creating scaffolds loaded with cells, but it necessitates precise deposition, droplet size, and viscosity adjustment [85]. LAB transfers cell-containing bioinks onto a substrate using laser radiation. High resolution is provided by this non-contact technique, which also develops bioinks with different viscosities effectively [86]. The fabrication of high-density scaffolds loaded with cells demands a complex arrangement of cell density and organization, a process where LAB proves highly effective. [85]. Extrusion printing is a widely used technique that enables the deposition of highly viscous bioinks, including the constructions that are packed with cells. This method forms continuous cylindrical filaments by extruding bioink using screw-driven, piston-driven, or pneumatic devices [87]. Although the extrusion approach has difficulties with cell survival because of shear stress, it enables high cell density and is appropriate for producing larger, more robust structures [85]. In SLA bioprinting, photosensitive bioinks are cured layer by layer with the application of light. It is especially helpful for designing complex micro- and nano-architectures [88]. SLA is appropriate for bioprinting scaffolds loaded with cells that need high resolution and structural integrity since it produces good cell viability because the light-curing process is non-contact [85]. These latest techniques manipulate cells and use magnetic or acoustic forces to create patterns without the need for physical contact. Acoustic bioprinting uses sound waves to create droplets loaded with cells, protecting them from shear stress, in contrast to magnetic bioprinting, which uses magnetic fields to assemble cells and is helpful for constructing multicellular 3D structures with less physical stress [89] [85]. Bioprinting for human tissue engineering involves three steps: pre-processing, processing, and post-processing. During pre-processing, human cells are isolated, cultured, and scaffold designs are created using CT or MRI for tissue modeling. In the processing phase, bioprinting techniques like inkjet, laser-assisted, and extrusion-based methods are employed to create 3D cell-laden scaffolds. Post-processing involves using bioreactors to ensure scaffold maturation under physiological conditions [90]. Bioinks play a crucial role in bioprinting. Structural bioinks provide mechanical stability, sacrificial bioinks create vascular-like channels, support bioinks aid softer materials, and functional bioinks enhance cellular processes. Bioinks are categorized into natural (e.g., collagen, alginate), synthetic (e.g., PCL, PEG), and composite types, balancing biocompatibility and mechanical strength.[85],[91],[92]. Cell-laden bioinks incorporate living cells to support growth and differentiation within scaffolds. Examples include collagen-alginate bioinks for bone and liver applications and GelMA, cross-linked with UV light, for bone and cartilage engineering. Hydrogel-based bioinks like alginate and fibrinogen provide cell-friendly environments. Stem cells, particularly MSCs and iPSCs, are central to regenerative medicine due to their differentiation potential. Bioprinting offers precise control over cell distribution and colony size, promoting lineage-specific differentiation. Growth factors like VEGF and BMPs are integrated into bioinks to guide cell behavior, enhancing applications in vascular and bone tissue engineering.[93],[94] [95]. 6. Scaffold design: Imitating Bone Microarchitecture for effective BTE In the BTE applications, the scaffold is the framework that provides the base for new bone regeneration. An ideal scaffold should closely mimic the native bone microarchitecture by means of replicating all the mechanical, chemical, and biological properties of bone to facilitate the cell growth, cell differentiation, and ultimately tissue regeneration. Herein, we report on key design features and parameters that scaffolds need to meet in order to become successful candidates for BTE (figure 3). 6.1. Biocompatibility and Bioactivity Biocompatibility ensures, that the scaffold does not evoke an adverse immune responses for immune rejection and does not cause any inflammation to their surrounding tissues[96], [97]. Bioactivity enhances the scaffold's ability to drive cell adhesion, proliferation, and differentiation[98]. Calcium phosphate ceramics, hydroxyapatite (HAp), and bio-glass are highly used for their osteoconductive properties which favor the formation of bone regeneration[99], [100], [101]. Biofunctionalization strategies such as peptide sequences increase cell–scaffold interaction, for example, through RGD motifs[102]. The presence of bioactive growth factors like BMP-2 , VEGF, TGF β, PDGF, IGF-1, FGFs allows scaffolds to give osteo-inductive and angiogenic support respectively, which is a very critical features in bone remodeling[103], [104], [105]. Nanostructured surfaces, which mimic the native extracellular matrix, have demonstrated higher bioactivity in acting not only as substrates for cell attachment but also as substrates for osteoblastic differentiation. The latest developments of biohybrid scaffolds based on combining synthetic polymers with biological components further enhance biocompatibility and bioactivity[106]. 6.2. Mechanical Properties and Structural Integrity Scaffolds of bone regeneration should be able to resist physiological loads without losing their structural integrity. Mechanical properties in native bones are hierarchical: the young modulus of cortical bone is higher (7-30 GPa) than that for trabecular bone (0.05-0.5 GPa) [107], [108]. The scaffold should provide a correspondingly wide range to avoid stress shielding and to allow transmission of the loads to newly forming tissue[109], [110]. For example, a study indicates that the composite, PCL-HA had a very good balance of mechanical strength and flexibility[111]. Gradient-layered scaffolds can be designed using finite element analysis to allow site-specific modulation of mechanical properties that will be ideal in complex load-bearing environments[112]. Other recent permutations include anisotropic scaffolds, designed to give directional mechanical properties, and auxetic scaffolds, which become thicker as they are stretched. The final two innovations serve to enhance mechanical compatibility with native bone tissue[113], [114]. 6.3. Porosity, Pore Size, and Interconnectivity The porosity of a scaffold ensures adequate space for cellular infiltration and vascularization that exchanges nutrients/waste. The optimal degree of porosity for bone in-growth has been reported between 60% – 80%. Therefore, pore sizes in the range of 100–400 µm have been more accepted in osteogenesis, while larger pores were favorable in angiogenesis around 500 µm[12], [115], [116]. Scaffolds with multiscale features are emerging as promising scaffold designs, such as those combining macropores to encourage vascularization with micropores for the attachment of osteoblasts. The degree of interconnectivity between individual pores is similarly important, serving to enhance the migration of cells and fluids within the scaffold tissue engineering construct. Advanced three-dimensional printing fabrication technologies offer unparalleled control over scaffold pore architecture, enabling controlled porosity and pore interconnectivity[115]. Figure 3: Representation of the key features and parameters that a 3-D scaffold needs to possess for the application in BTE. 6.4 Biodegradability and Regulated Degradation Scaffolds degrade at a rate which must be matched to the tissue regeneration process. The natural biopolymers such as chitosan, alginate, gelatin, collagens, hyaluronic acid and the synthetic polymers such as PLA, PGA, PCL, PLGA had been widely used for scaffold fabrication for their biocompatible and biodegradable properties, these polymers controls the degradation rate of the scaffold based on their stiffness and the crosslinking strategy applied[117]. PLA and PCL, materials usually utilized, degraded hydrolytically and gave non-toxic by-products[118]. A too slow degradation rate will not support the development of the tissues whereas fast degradation will lead to the failure of scaffolds and cause an inflammation to the surrounding of the regenerating tissues. Cross-linked polymers and multi-phase composites have tunable degradation rates guaranteeing stability for a long period of time[119]. To this end, novel approaches, such as polymer degradation by enzymatic activation or pH sensitivity, are allowing a more controlled degradation of scaffolds nowadays. Again, ion-releasing bio-ceramics like β-TCP will create a favorable environment for osteogenesis, assisting in bone remodeling when degrading[120], [121]. 6.5. Vascularization and Angiogenesis Biologically, vascularization is considered one of the critical factors in the success of bone regeneration since cell survival relies on the provision of oxygen and nutrients. New pre-vascularized scaffolds developed by the co-culture of endothelial cells with osteoblast cells are becoming popular because they allow rapid vascularization[122]. Additionally, the incorporation in scaffolds of microspheres loaded with VEGF has given fairly good results for angiogenesis enhancement[123]. Advanced bioprinting provides microvascular networks that ideally mimic the native vasculature and allow for efficient oxygen diffusion within the scaffold[124]. Other approaches, such as hypoxia preconditioning, give promise of enhanced angiogenesis through increased VEGF expression[125]. 6.6 Surface Properties and Functionalization Surface properties of the scaffolds are one of the major variables influencing cell behavior. Nano-rough surfaces, which mimic native bone ECM, refer to enhanced adhesion and differentiation of osteoblasts[126]. In this direction, functionalization techniques like plasma treatment, silanisation, and coating with bioactive molecules enhance scaffold wettability and bioactivity[127], [128], [129]. The current research has been directed towards stimulus-responsive scaffolds, which on demand, can deliver therapeutic agents due to changes in pH, temperature, or light. Such advanced scaffolds offer new possibilities for both targeted drug delivery and enhanced tissue regeneration[130]. 6.7. Customization through 3-D Printing and Computational Modeling It has been demonstrated that patient-specific scaffolds fabricated from computed tomographic or magnetic resonance imaging scans allow for more precise anatomical reconstruction of complex bony defects[131]. By printing scaffolds using multiple materials, it is possible to obtain rigid and flexible regions in the same scaffold, mimicking the natural gradient in properties within the tissues of bone. Different computational modelling tools, like finite element analysis and topology optimization, made the prediction of scaffold performances due to mechanical stress possible in ensuring scaffold durability[132]. It also extends into recent developments allowing in-silico simulations to study cell-scaffold interactions while reducing dependence on extensive cell culture-based experiments in-vitro[133]. 7. Clinical applications and future directions Apart from Bone Tissue Engineering, there are wide range of clinical approaches of the 3-dimensional scaffolds (figure 4). Mesenchymal stem cells (MSCs), especially adipose-derived stem cells (ADSC) and bone marrow-derived stem cells (BMSC), can be incorporated into the three-dimensional scaffolds to aid bone regeneration. These stem cells encourage osteogenic development when integrated into bio-printed scaffolds, improving the regeneration and repair of bone tissue. Various bioprinting methods, including extrusion, inkjet, and laser-assisted, are used according to the required scaffold properties, print accuracy, and cell viability [134]. Biosensor functionality can be supported by bioinks made using sophisticated 3-D bioprinting processes. These biosensors, which are integrated into bioprinted structures, may be used to track physiological parameters or promote electrical transmission in tissues, such as in the case of heart tissue regeneration [135]. In large structures like livers or kidneys, which depend on an integrated circulatory network to operate correctly, vascularization procedures, for example, are essential for maintaining cell viability. Additionally promising for applications in skin, heart, and bone tissue is the combination of stem cells and growth factors in bioprinted scaffolds, particularly in the development of intricate, multi-layered tissue systems[94]. By combining 3D bioprinting with microfluidic devices, organ-on-a-chip technology is advanced and in-vitro models that more closely resemble human physiology can be developed. These devices are more effective in drug screening and illness research because bioprinting makes it possible to precisely insert scaffolds loaded with cells inside of them [136]. The target tissues, this technique is particularly helpful for tissues like the liver or skin that require uniform cell distribution because cellular function depends on even spacing and steady access to nutrients. Circulation-assisted bioprinting overcomes an inkjet bioprinting constraint and is a step toward producing more complicated, cell-dense tissues. For sensitive applications, future research may concentrate on enhancing control over flow rates, reducing shear stress on cells, and optimizing circulation mechanisms [91]. It is expected that using AI to create intricate bioprinting designs and optimize print settings will increase the accuracy and productivity of bioprinting [137]. Microbial consortia are formed by bioprinting microorganisms in particular spatial patterns, which enables the regulated synthesis of bioproducts like biofuels and antibiotics. This configuration facilitates communication and collaboration among many microorganisms, which is beneficial for biosynthesis and bioprocessing. Additionally, microbial 3D bioprinting is being used to create "living" responsive devices, such wearable biosensors that are able to detect changes in their surroundings. These gadgets could be used in customized medicine to track or react to physiological changes [138]. Bioprinting approaches improve osteogenic differentiation, the process by which stem cells develop into bone cells, by combining various materials in scaffolds. Growth factors or bioactive compounds can be incorporated into the scaffolds to guide the development of stem cells into osteoblasts, or cells that produce bones. Stronger, more durable bone-like structures can be produced thanks to multimaterial scaffolds that are designed to enhance the construct's mechanical qualities. In orthopedic therapies, these characteristics are essential for load-bearing applications [134]. Bioprinting can address donor compatibility and immunological rejection by customizing organ and tissue structures for each patient. Transplant recipients may benefit from customized solutions offered by personalized bioprinted organs, improving results. A primary goal is the development of increasingly complex bioinks that promote tissue function and cell viability. The ideal bioinks would include signaling molecules that direct the body's integration and growth of new tissue in addition to cells. Enhancing techniques for vascular network formation within bioprinted tissues is the goal of future research since it is crucial for supporting larger organ architectures. This involves methods for creating blood artery networks that can be bioprinted to support the flow of nutrients and oxygen in thicker tissues[139]. Figure 4: Illustration of the clinical applications and prospective advancements of the 3D-printed scaffolds in tissue engineering. 8. Conclusion: Finally, the incorporation of various 3D printing techniques supports a remarkable enhancement in bone tissue engineering. SLS, SLA, and bioprinting are among the techniques used to fabricate scaffolds for bone regeneration with precision. These techniques have facilitated the fabrication of such complex, biomimetic scaffolds, accurately reproducing optimum microarchitectural features of natural bone, including pore size, interconnectivity, and mechanical strength. To further enhance the performance of 3D-printed scaffolds, bioactive materials of both natural origin and synthetic, as well as composite materials, have been incorporated. These biomaterials promote stem cell differentiation, angiogenesis, immune response modification, and provide a scaffold that is conductive for osteoinductive events necessary for appropriate bone repair. Furthermore, the engineering of bioprinting with cell-laden scaffolds is a significant accomplishment because one can directly embed both growth factors and stem cells into the scaffolding material for more efficient and controlled repair of bone. Advanced scaffold design, based on phytocompounds, holds promise for improving scaffold bioactivity as bone tissue engineering approaches the clinic. The natural bioactive properties of the studied phytocompounds have been reported to induce bone tissue regeneration, improve cellular adhesion, and enhance the process of osteogenesis. 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